Thrombolytic agents and methods of treatment for thrombosis

ABSTRACT

The foregoing invention relates to a new microbubble preparation and thrombolytic therapy which relies on microbubbles and ultrasound for its lytic activity. The pharmaceutical composition of the invention comprises a liquid solution of microbubbles with an internal atmosphere enhanced with the perfluorocarbon gas which cavitate in the presence of an ultrasound field following intravenous injection or infusion of said composition into said host. For thrombolysis the area of a thrombus is exposed to an ultrasound field in the presence of the microbubbles and significant lysis is experienced. The method and pharmaceutical composition of the invention exhibit thrombolytic properties similar to those of other thrombolytic agents such as urokinase and are less toxic and are clot specific in that they do not introduce a systemic lytic state to a said animal.

FIELD OF THE INVENTION

[0001] This invention relates to a new and improved pharmaceuticalcomposition and method for treating thrombosis in animals. The methodsand composition of the invention can be used as an anticoagulant therapyto induce thrombolysis and to relieve trauma associated with obstructionof smaller vessels.

BACKGROUND OF THE INVENTION

[0002] Thrombosis, the formation and development of a blood clot orthrombus within the vascular system, while a life saving process when itoccurs during a hemorrhage, can be life threatening when it occurs atany other time. The thrombus can block a vessel and stop blood supply toan organ or other body part. If detached, the thrombus can become anembolus and occlude a vessel distant from the original site.

[0003] In the healthy person there is a balance between clot formation(thrombosis) which is needed to minimize blood loss and to repair bloodvessels, and clot lysis (fibronolysis) which maintains the patency ofblood vessels. When thrombosis occurs without concomitant fibronolysiseffects can lead to strokes.

[0004] Traditional thrombolytic agents used are not clot specific andwhile they do break up the thrombus and facilitate fibronolysis theyalso put the patient at significant risk as all clotting is inhibitedand a patient could bleed to death from a small abrasion elsewhere.Current thrombolytic agents include streptokinase which is derived fromBeta-hemolytic streptococci. When combined with plasminogen,streptokinase catalyzes the conversion of plasminogen to plasmin, theenzyme responsible for clot dissolution in the body. Three majorproblems encountered with the use of streptokinase therapy include itssystemic lytic effects coupled with a long half life. Because theanticoagulant activity of streptokinase is indiscriminent (non clotspecific) and prolonged (half life 10-18 minutes), bleeding is a commoncomplication which must be carefully monitored during 12 hours followingimmediately after administration. Further because streptokinase is abacterial protein, it is strongly antigenic and can produce a variety ofallergic reactions including anaphylaxis, particularly when administeredto a patient who has previously received streptokinase therapy or whohas had a recent streptococcal infection.

[0005] Another popular agent for use in treatment of thrombosis isurokinase, an enzyme protein secreted by the parenchyma cells of thehuman kidney. It acts to direct activation of plasminogen to formplasmin. This is different from streptokinase which first forms acomplex with plasminogen to activate plasmin to dissolve the clot.Urokinase is also non clot specific (activates circulating non clotbound plasminogen as well as clot bound plasminogen) but has a shorterhalf life than streptokinase. Its administration is associated withfewer bleeding complications despite the fact that a systemic lyticstate is also produced. Urokinase is produced by the kidney and as suchit is not antigenic and well suited for use if subsequent thrombolytictherapy is needed. The major problem with urokinase is that it isdifficult and expensive to produce precluding its extensive clinicaluse.

[0006] The most recently developed drug in treating of thrombolysis isrecombinant tissue plasminogen activator. Approved by the FDA inNovember of 1987, tissue plasminogen activator (t-PA) is a naturallyoccurring enzyme (thus non antigenic) that is clot specific and has avery short half life (3-5 minutes). It converts plasminogen to plasminafter binding to the fibrin-containing clot. This clot specificityresults in an increased concentration and activity of plasmin at thesite of the clot, where it is needed. This characteristic of t-PAprevents the induction of the systemic lytic state that occurs withstreptokinase and urokinase activity. However the results of studiescomparing the streptokinase and t-PA show similar incidences of bleedingfollowing administration. Successful gene cloning has made sufficientquantities of t-PA available for clinical use, however, the recombinanttechnology necessary for its production have also resulted in aprohibitive cost. As can be seen a need in the art exists for athrombolysis therapy which is clot specific, which does not induce asystemic lytic state and which is inexpensive and non antigenic topatients.

SUMMARY OF THE INVENTION

[0007] According to the invention a thrombolytic therapy is providedwhich is site-specific and non-antigenic. The therapy involves the useof a pharmaceutical composition which comprises microbubbles of adiameter of about 0.1 to 10 microns, the interior of which has beenenhanced with an insoluble gas such as fluorocarbon gas, helium orsulfur hexafluoride and which gas is encapsulated in a protein-coatedshell. The invention uses agents and methods traditionally used inultrasound imaging and as such provides a means for visualization of theclot as it is being lysed. Quite unexpectedly it was found that theinsoluble gas microspheres of the invention act themselves as athrombolytic agent in the presence of an ultrasound field and work aswell as traditional thrombolytic agents such as urokinase.

DETAILED DESCRIPTION OF THE INVENTION

[0008] Ultrasonic imaging has long been used as a diagnostic tool to aidin therapeutic procedures. It is based on the principle that waves ofsound energy can be focused upon an area of interest and reflected toproduce an image. Generally an ultrasonic transducer is placed on a bodysurface overlying the area to be imaged and ultrasonic energy, producedby generating and receiving sound waves, is transmitted. The ultrasonicenergy is reflected back to the transducer where it is translated intoan ultrasonic image. The amount of characteristics of the reflectedenergy depend upon the acoustic properties of the tissues, and contrastagents which are echogenic are preferably used to create ultrasonicenergy in the area of interest and improve the imaging received. For adiscussion of contrast echographic instrumentation, see, DeJong and,“Acoustic Properties of Ultrasound Contrast Agents”, CIP-GEGEVENSKONINKLIJKE BIBLIOTHEEK, DENHAG (1993), pp. 120 et seq.

[0009] Contrast echocardiography has been used to delineate intracardiacstructures, assess valvular competence, and demonstrate intracardiacshunts. Myocardial contrast echocardiography (MCE) has been used tomeasure coronary blood flow reserve in humans. MCE has been found to bea safe and useful technique for evaluating relative changes inmyocardial perfusion and delineating areas at risk.

[0010] Ultrasonic vibration has also been used in the medical field toincrease the absorption of various medicaments. For example in JapanesePatent Kokai number 115591/1977 discloses that percutaneous absorptionof a medicament is enhanced by applying an ultrasound vibration. U.S.Pat. Nos. 4,953,565 and 5,007,438 also disclose a technique ofpercutaneous absorption of medicaments by the aid of ultrasonicvibration. U.S. Pat. No. 5,315,998 discloses a booster for drug therapycomprising microbubbles in combination ultrasonic energy to allow themedicament to diffuse and penetrate at the site of interest.

[0011] Quite surprisingly applicant has demonstrated that a microbubblecomposition in combination with ultrasound therapy can act as athrombolytic medicament causing clot lysis at the site of a thrombus. Inthe presence of ultrasound the microbubbles themselves act as amedicament and are as effective as traditional thrombolytic agents suchas urokinase or t-PA. The pharmaceutical composition of the inventioncomprises a liquid containing microbubbles of an insoluble gas having adiameter of 0.1 to 10 microns. The microbubbles are formed by entrappingmicrospheres of a gas into a liquid. The microbubbles are made ofvarious gases preferably inert gases as xenon, krypton, argon, neon,helium, or fluorocarbon gases. The liquid includes any liquid which canform microbubbles. Generally any inert gas can be used. It must begaseous at body temperature and be nontoxic. The gas must also formstable microbubbles of average size of between about 0.1 and 10 micronsin diameter when the pharmaceutical composition is sonicated to formmicrobubbles. Generally perfluorocarbon gases such as perfluoromethane,perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane arepreferred. Of these gases, perfluoropropane and perfluorobutane areespecially preferred because of their demonstrated safety forintraocular injection in humans. They have been used in human studiesfor intraocular injections to stabilize retinal detachments (Wong andThompson, Opthamology 95:609-613). Treatment with intraocularperfluoropropane is considered to be the standard of care for treatmentof this disorder. The gases must also have a diffusion coefficient andblood solubility lower than nitrogen or oxygen which diffuse once in theinternal atmosphere of the blood vessel.

[0012] Other inert gases such as sulfur hexafluoride are also useful inthe invention provided they have a diffusion coefficient and bloodsolubility lower than nitrogen or oxygen. The agent of the invention isformulated in a pharmaceutically effective dosage form for peripheraladministration to the host in conjunction with ultrasound therapy.Generally such host is a human host, although other mammalian hosts suchas canine or equine can also be subject to this thrombolytic therapy.

[0013] In a preferred embodiment the pharmaceutical liquid compositionof the invention uses a liquid wherein the microbubbles are stabilizedby a filmogenic denaturable protein coating. Suitable proteins includenaturally occurring proteins such as albumin, human gamma globulin,human apatransferin, Betalactose and urease. The invention preferablyemploys a naturally occurring protein but synthetic proteins may also beused. Particularly preferred is human serum albumin.

[0014] It is also preferred to use an aqueous solution containing amixture of a pharmaceutically accepted saccharide e.g., dextrose, incombination with the earlier described protein. In a most preferredembodiment the pharmaceutical liquid composition of the invention is thesonicated mixture of commercially available albumin (human), U.S.P.solution (generally supplied as 5% or 25% by weight sterile aqueoussolutions), and commercially available dextrose, U.S.P. for intravenousadministration. The mixture is sonicated under ambient conditions i.e.room air temperature and pressure and is perfused with an insoluble gas(99.9% by weight) during sonication.

[0015] In a most preferred embodiment the pharmaceutical liquidcomposition includes a two-fold to eight-fold dilution of 5% to 50% byweight of dextrose and a 2% to 10% by weight of human serum albumin.Exemplary of other saccharide solutions of the invention are aqueousmonosaccharide solution (e.g. having the formula 6CH6012 such as thehexos sugars, dextrose or fructose or mixtures thereof), aqueousdisaccharide solution (e.g. having a formula C₁₂H₂₂O₁₁ such as sucrose,lactose or maltose or mixtures thereof), or aqueous polysaccharidesolution (e.g. soluble starches having the formula C₆H₁₀O₅(n) wherein nis a whole number integer between 20 and about 200 such as amylase ordextran or mixtures thereof.

[0016] The microbubbles are formed by sonication, typically with asonicating horn. Sonication by ultrasonic energy causes cavitationwithin the dextrose albumin solution at sites of particulate matter orgas in the fluid. These cavitation sites eventually resonate and producesmall microbubbles (about 7 microns in size) which are non-collapsingand stable. In general, sonication conditions which produceconcentrations of greater than about 4×10⁸m of between about 5 and about6 micron microbubbles are preferred. Generally the mixture will besonicated for about 80 seconds, while being perfused with an insolublegas.

[0017] A second method of preparation includes hand agitating 15±2 ml ofsonicated dextrose albumin with 8±2 ml of perfluorocarbon gas prior tosonication. Sonication then proceeds for 80±5 seconds. Generally thepharmaceutical liquid composition is injected into the area of thethrombosis or close thereto and then ultrasound is applied.

[0018] These microbubble sizes are particularly ideal since amicrobubble must have a mean diameter of less than 10 microns andgreater than 0.1 to be sufficient for transpulminary passage, and mustbe stable enough to prevent significant diffusion of gases within themicrobubble following intravenous injection and during transit to thethrombosis site. The method preferred for practicing the anti thrombosistherapy of the invention involves obtaining a pharmaceutical liquidagent of the invention, introducing said agent into a host byintravenous injection, intravenously (i.v. infusion), percutaneously orintramuscularly. Injection is such that the area of the thrombus isperfused with the pharmaceutical composition. Next ultrasound is appliedthereto using a suitable Doppler or ultrasound echo apparatus so thatthe field of ultrasound encompasses the thrombus. The ultrasound signalactivates the microbubbles so that the microbubbles themselves act as athrombolytic agent.

[0019] The desired ultrasound is applied by conventional ultrasonicdevices which can supply an ultrasonic signal of 20 Khz to several Mhzand is generally applied from about 3 to about 5 Mhz.

[0020] In the most preferred embodiment the agent of the invention is aperfluorocarbon enhanced sonicated dextrose albumin solution comprisedof a sonicated three-fold dilution of 5% human serum albumin with 5%dextrose. During sonication, the solution is perfused withperfluorocarbon gas for about 80 seconds which lowers the solubility anddifusivity of the microbubble gas. The resulting microbubbles areconcentrated at room temperature for at least about 120±5 minuteswherein the excess solution settles in the sonicating syringe. Theexcess solution is expelled and the concentrated microbubbles aretransferred to a sterile syringe and injected parenterally into amammal, near the site of the thrombus.

[0021] Methods of ultrasonic imaging in which microbubbles formed bysonicating an aqueous protein solution are injected into a mammal toalter the acoustic properties of a predetermined area which is thenultrasonically scanned to obtain an image for use in medical proceduresis well known. For example see U.S. Pat. Nos. 4,572,203, 4,718,433 and4,774,958, the contents of each of which are incorporated herein byreference.

[0022] It is the use of these types of contrast agents as apharmaceutical composition and application of ultrasound as an antithrombosis therapy that is the novel improvement of this invention.Blood clots when treated with the microbubble composition and therapy ofthis invention were shown to decrease in size by a percentage equal tothat of traditional thrombolytic agents such as urokinase. According tothe invention, it was shown that treatment with decafluorobutanesonicated dextrose albumin microbubbles and subsequent application ofultrasound resulted in a higher percentage of clot reduction thantreatment with urokinase alone. The combination of perfluorocarbonenhanced sonicated dextrose albumin microbubbles (PESDA) and ultrasoundresulted in increased clot lysis from that of ultrasound alone or overuse of PESDA alone.

[0023] This is particularly significant as the microbubbleanti-thrombosis therapy can reduce any toxic effects of persons whocannot otherwise use traditional thrombolytic agents such as urokinase.According to the invention the thrombosis can be treated simply withultrasound in combination with a microbubble pharmaceutical compositionof the invention and the protein substance such as human serum albuminis easily metabolized within the body and excreted outside and hence isnot harmful to the human body. Further gas trapped within themicrobubbles is extremely small and is easily dissolved in blood fluid,perfluoropropane and perfluorobutane have long been known to be safe inhumans. Both have been used in humans for intra ocular injections tostabilize retinal detachments. Wong and Thompson, Opthalmology95:609-613. Thus the anti thrombosis agents of the invention areextremely safe and nontoxic for patients.

[0024] The following examples are for illustration purposes only and arenot intended to limit this invention in any way. These examplesdemonstrate the effect of the pharmaceutical compositions and therapy ofthe invention. In all the following examples, all parts and percentagesare by weight unless otherwise, all dilutions are by volume.

EXAMPLES Preparation of Anti Thrombosis Pharmaceutical Agent

[0025] Albumin (human) USP, 5% solution (hereinafter referred to as“albumin”) and dextrose USP, 5% solution (hereinafter referred to as“dextrose”) were obtained from a commercial source. The sonicatingsystem used for sonication was a Heat System Ultrasonic Processor ModelXL2020 (Heat Systems Inc., Farmingdale, N.Y.). The ½ inch horntransducer was a resonating piezoelectric device. The ½ inch sonicatinghorn tip was sterilized prior to each sonication.

Sonication of Samples

[0026] Sixteen milliliter aliquots of albumin diluted 1:3 with dextrosewere drawn up into a 35 cc “Monoject” syringe (Becton Dickinson andCompany, Rutherford, N.J.) and sonicated for 80±1 seconds. The“Leur-Lok” of the 35 milliliter syringe was then attached to a stopcock.After mixing the dextrose albumin solution by hand for about 7 to about10 seconds, the plunger was removed from the top of the syringe. Thesterile sonicating horn was then lowered into the open end of thesyringe until at the surface of the albumin-dextrose solution. Thesolution was placed at the horn tip and manually held at this positionwhile continuously sonicating at a frequency of 20,000 Hz and a poweroutput of 210 W for 80±1 seconds to form a stable microbubble solution.

Gas Perfusion of Samples

[0027] A second method of preparation includes hand agitating 15±2 ml ofsonicated dextrose albumin with 8±2 ml of perfluorocarbon gas prior tosonication. Sonication then proceeds for 80±5 seconds. Generally thepharmaceutical liquid composition is injected into the area of thethrombosis or close thereto and then ultrasound is applied.

[0028] The dextrose albumin mixture was exposed to eitherperfluoropropane or perfluorobutane gas (Commercial Grade, 99.9% byweight) by hand agitating 15±2 ml of sonicated dextrose albumin with 8±2ml of perfluorocarbon gas prior to sonication. Theperfluorocarbon/dextrose-albumin mixture was then sonicated for 80±5seconds. The total volume of perfluorocarbon-enhanced sonicated dextrosealbumin produced with this formulation was 25±2 milliliters. Thesesamples were then used for intravenous injection.

Microbubble Analysis

[0029] Microbubble size and purity was determined using hemocytometry.Microscopic inspection of the microbubbles was performed to determine ifany coalescent microbubbles were present in the solution. Microbubbleconcentration was determined using a Coulter Counter. The antithrombosis pharmaceutical agent was rejected for use if any of thefollowing conditions are present: the mean microbubble size was 4.0 to6.0 microns; coalesced microbubbles or strands were detected by lightmicroscopy; or the mean microbubble concentration was less than 0.8×10⁹or greater than 1.5×10⁹ microbubble/milliliter. The sample was alsorejected if the number of microbubbles greater than 10 microns in thesample was greater than 4%.

[0030] All samples were stored in 35 milliliter syringes until time ofinjection. All solutions were given within 36 hours of production. Allsamples were prepared in a laminar flow hood.

Example 1 In Vitro Clot Lysis Using PESDA and Ultrasound

[0031] The pharmaceutical compositions and method of the invention wereshown to reduce the size of blood clots according to the following invitro protocol. The protocol is known in the art and is predictive ofsuccess in vivo Sehgal, “Ultrasound-Assisted Thrombolysis”,Invest-Radiol., October 1993, Vol. 28, No. 10:939-43. 2 ml aliquots offreshly drawn whole blood were placed into a 10 cc plunger invertedsyringes. The blood was then incubated for 2 hours at 37° C. Afterincubated, the syringes were removed from the water bath and left atroom temperature until treatment. Upon treatment the serum was decantedfrom the clot by pouring the contents of the syringe over a wire meshscreen. The clot was then dried by rolling in the screen and blotting.The clot was then weighed and placed back into the syringe with lyticfluid (microbubble pharmaceutical composition of the invention). Sampleswithout treatment were incubated at 37° C. in a water bath for 20minutes. Samples with treatment involved placement of the ultrasoundhorn approximately 2 ml in solution and ultrasound was applied for 2minutes. After 2 minutes the clot was incubated for 18 minutes at 37° C.Again the fluid was decanted, the clot was rolled and blotted on thebottom of the screen to dry and the clot was weighed subsequent totherapy.

[0032] Several experiments were run using this protocol and the resultsare shown in the following tables. Experiment #1 With Ultrasound WithoutUltrasound % clot lysis % clot lysis Sample n Average Sample n Averagesaline 4  7.4 saline 4 8.7 urokinase 4 46.3 urokinase 5 17.9  PESDA 415.3 PESDA 4 3.1

[0033] Experiment #2 With Ultrasound Without Ultrasound % clot lysis %clot lysis Sample n Average Sample n Average saline 4 33.3 saline 3 4.1urokinase 4 54.9 urokinase 4 12.4  PESDA 6 58.7 PESDA 4 3.1

[0034] Experiment #3 With Ultrasound Without Ultrasound % clot lysis %clot lysis Sample n Average Sample n Average saline 4 10.9 saline 4 7.8urokinase 4 45.1 urokinase 4 17.7  PESDA 4 50.9 PESDA 4 4.1

[0035] Experiments 1, 2 and 3 combined With Ultrasound WithoutUltrasound % clot lysis % clot lysis Sample n Average Sample n Averagesaline 12 12.9 saline 11 6.9 urokinase 12 48.8 urokinase 13 16   PESDA14 44.0 PESDA 12 3.4

[0036] As can be seen from the foregoing tables, when all data iscombined with over 10 separate experiments, ultrasound in combinationwith perfluorobutane enhanced, sonicated dextrose albumin microspheresdemonstrated an average percent clot lysis that was approximately equalto that which resulted from urokinase in combination with ultrasound.

[0037] As can be seen quite unexpectedly, in the presence of ultrasound,PESDA microbubbles work as a thrombolytic agent to reduce the size of athrombous at a level which rivals that of traditional thrombolyticagents such as urokinase.

Example 2 (Prophetic)

[0038] For humans the anti thrombosis therapy includes doses of theliquid pharmaceutical composition, PESDA, from about as small as 0.0025up to 0.1 ml/kg given depending on the ultrasonic procedure used. Thecontrast agent is given by peripheral intravenous infusion over about1-25 minutes (the dose range is patient specific. Large patients mayrequire slightly higher doses to produce equivalent thrombolysis).Generally in one protocol a patient will receive a 0.01 ml/kg ofperfluorocarbon enhanced sonicated dextrose albumin or 0.0015 ml/kgperfluorobutane sonicated dextrose albumin as the initial injection. Ifthis fails to produce significant clot lysis, the dose could then bedoubled. Dosing protocols would be similar to those used for ultrasoundimaging and are disclosed in Wyman, Arthur E. “Principles and Practiceof Echocardiography”, Lee & Febiger, Malvern, Pa. (1994 2nd Edition).Any ultrasound device can be used including the commercially availableHewlett Packard Sonus 1500 Phased Ray Imaging System (Hewlett Packard,Andover Mass.). The patient is exposed to ultrasound for a timesufficient to experience significant clot lysis and generally will befrom about 1 to about 25 minutes. Thrombolysis can be monitored byviewing with conventional angiography, using radiographic dyes, or otheraccepted methods.

What is claimed is:
 1. A method of treating thrombosis in animalscomprising the steps of: introducing a pharmaceutical composition to athrombus site by intravenous injection, said pharmaceutical compositioncomprising a plurality of gas filled microbubbles with a diameter offrom about 0.1 to 10 microns and a pharmaceutically acceptable carrier,and thereafter; applying ultrasound to said site.
 2. The method of claim1 wherein said gas is an insoluble gas.
 3. The method of claim 1 whereinsaid microbubbles are protein coated.
 4. The method of claim 1 whereinsaid carrier is a 5% solution of dextrose.
 5. The method of claim 3wherein said protein coated microspheres are albumin coatedmicrospheres.
 6. The method of claim 2 wherein said insoluble gas isselected from the group consisting of perfluoromethane, perfluoroethane,perfluoropropane, perfluorobutane and perfluoropentane.
 7. The method ofclaim 6 wherein said perfluorocarbon gas is perfluorobutane.
 8. Themethod of claim 6 wherein said perfluorocarbon gas is perfluoropropane.9. The method of claim 1 further comprising the following steps: mixingan aqueous solution comprising 2% to about 10% by weight of human serumalbumin diluted about two fold to about eight fold with 5% to 50% byweight dextrose; and exposing said solution to a sonication horn togenerate stable microbubbles from about 0.1 to 10 microns in diameter,to create said pharmaceutical composition.
 10. The method of claim 7wherein said dilution of albumin with dextrose is a 3-fold dilution. 11.The method of claim 7 wherein said human serum albumin is a 5% by weightsolution.
 12. The method of claim 7 wherein said dextrose is a 5% byweight solution.
 13. A method for treating thrombosis in animalscomprising: (a) obtaining a pharmaceutical composition which consistsessentially of: a solution of stable microbubbles aproximately 0.1 to 10microns in diameter, and a pharmaceutically acceptable carrier; (b)introducing said pharmaceutical composition to said thrombus; and (c)exposing said pharmaceutical composition and said thrombus to anultrasound field for a time sufficient to lyse said thrombus.
 14. Themethod of claim 13 wherein said step of introducing said agent to saidthrombus is by intravenous injection.
 15. The method of claim 13 whereinsaid dextrose is a 5% solution.
 16. The method of claim 13 wherein saidprotein coated microbubbles are albumin coated microspheres.
 17. Themethod of claim 13 wherein said perfluorocarbon gas is selected from thegroup consisting of perfluoromethane, perfluoroethane, perfluoropropane,perfluorobutane and perfluoropentane.
 18. The method of claim 17 whereinsaid perfluorocarbon gas is perfluorobutane.
 19. The method of claim 17wherein said perfluorocarbon gas is perfluoropropane.
 20. The method ofclaim 13 wherein said further comprising the following steps: mixing anaqueous solution comprising 2% to about 10% by weight of human serumalbumin diluted about two fold to about eight fold with 5% to 50% byweight dextrose; and exposing said solution to a sonication horn tocreate cavitation at particulate sites in said solution generatingstable microspheres from about 0.1 to 10 microns in diameter, to formsaid pharmaceutical composition.
 21. The method of claim 20 wherein saiddilution of albumin with dextrose is a 3-fold dilution.
 22. The methodof claim 18 wherein said human serum albumin is a 5% by weight solution.23. The method of claim 20 wherein said dextrose is a 5% by weightsolution.
 24. A method for lysing a thrombus in an animal comprising:introducing a pharmaceutical composition to said animal by intravenousinjection near a thrombus site, said pharmaceutical compositioncomprising a microbubble ultrasound contrast agent, and thereafter;applying ultrasound to said site.
 25. A method for lysing a thrombus inan animal comprising: introducing a pharmaceutical composition to saidanimal by intravenous injection near a thrombus site, saidpharmaceutical composition comprising a microbubble ultrasound contrastagent, and thereafter; applying ultrasound to said site.